Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.     

Vitamin D receptor gene polymorphism is associated with serum total and ionized calcium concentration

Restriction fragment length polymorphisms of the vitamin D receptor gene have recently been reported to be associated with changes in bone mineral density. Alterations in systemic calcium balance and Ca-regulating hormones such as 1,25(OH)2 vitamin D3 and parathyroid hormone have been demonstrated in essential hypertension. We investigated the relationship between polymorphisms of the vitamin D receptor gene and systemic Ca metabolism in patients with essential hypertension and in normotensives. We compared 147 subjects with essential hypertension and 100 normotensive control subjects. The genotype distribution and derived allele frequencies for the vitamin D receptor gene were similar in the two groups (genotype bb/Bb/BB and allele B/b: 60.1/32.6/7.2 and 0.24/0.76 in hypertensives vs. 56.0/36.0/8.0 and 0.26/0.74 in normotensive subjects). Serum concentrations of total Ca in the bb, Bb, and BB groups were, respectively, 4.5+/-0.3 vs. 4.5+/-0.4 vs. 4.4+/-0.5 mmol/l in normotensives and 4.6+/-0.3 vs. 4.6+/-0.4 vs. 4.4+/-0.5 mmol/l in hypertensives. Ionized Ca levels were 1.17+/-0.04 vs. 1.16+/-0.04 vs. 1.15+/-0.04 mmol/l in normotensives and 1.16+/-0.04 vs. 1.16+/-0.04 vs. 1.14+/-0.05 mmol/l in hypertensives, respectively. These results indicate that the BB genotype of the vitamin D receptor gene is associated with lower serum Ca levels but is not a useful predictive marker for the development of essential hypertension in Japanese subjects.     

Text comprehension and individual differences in working memory: the different contributions of the storage and the retrieval processing

The present study addressed the relationship between an individual difference in the reading span test (RST) and the performance in text comprehension with respect to storage and retrieval systems. In Experiment 1, an effect of the serial recall task on performance in text comprehension was compared between high and low RST score groups. In Experiment 2, an effect of the word fluency task on performance in text comprehension of two groups was investigated. The results of both experiments showed that the performance in text comprehension of the low RST score group was impaired when the serial recall task was the secondary task; in contrast, the performance of the high RST score group was influenced when the word fluency task was used. The results suggested that the high RST score group comprehended text not only by using a temporary storage system but also by using a retrieval system efficiently.     

Development of early neutropenic fever, with or without bacterial infection, is still a significant complication after reduced-intensity stem cell transplantation.

Little information is available on the clinical characteristics of infectious complications that occur in the early period after reduced-intensity stem cell transplantation (RIST). We retrospectively investigated the clinical features of neutropenic fever and infectious episodes within 30 days after RIST in 76 patients who had received fluoroquinolones as part of their antibacterial prophylaxis. Preparative regimens included cladribine 0.66 mg/kg or fludarabine 180 mg/m2 plus busulfan 8 mg/kg. All but 1 patient survived 30 days after transplantation, and 75 patients (99%) became neutropenic within a median duration of 9 days. Neutropenic fever was observed in 29 patients (38%), and bacterial infection was confirmed in 15 (20%) of these, including bacteremia (n = 13), bacteremia plus pneumonia (n = 1), and urinary tract infection (n = 1). The causative organisms were gram-positive (n = 9) and gram-negative organisms (n = 7), with a mortality rate of 6%. Neither viral nor fungal infection was documented. Multivariate analysis showed that the presence of neutropenia at the initiation of preparative regimens was an independent risk factor for subsequent documented bacterial infections (P =.026; 95% confidence interval, 1.25-35.1). We conclude that neutropenic fever and bacteremia remain common complications in RIST.     

Functional evaluation of lung by Xe-133 lung ventilation scintigraphy before and after lung volume reduction surgery (LVRS) in patients with pulmonary emphysema

We evaluated the respiratory functions of patients with pulmonary emphysema who underwent lung volume reduction surgery (LVRS) by the mean transit time (MTT) with Xe-133 lung ventilation scintigraphy, forced expiration volume in 1 sec (FEV1.0), residual volume (RV), distance walked in 6 min (6-min walk), and the Hugh-Jones classification (H-J classification) before and after LVRS. In 69 patients with pulmonary emphysema (62 men, 7 women; age range, 47-75 years; mean age, 65.4 years +/- 6.1, preoperative H-J classification, III (two were II)-V) who underwent LVRS, all preoperative and postoperative parameters (MTT 3 weeks after LVRS and the others 3 months after LVRS) were judged statistically by the Wilcoxon signed-ranks test and Odds ratio. Every postoperative parameter was improved with a significant difference (P < 0.05) compared to preoperative parameters. MTT at 3 weeks after LVRS was not associated with %FEV1.0 and the H-J classification at 3 months after LVRS, but was associated with RV and a 6-min walk at 3 months after LVRS. MTT was useful for the clinical evalution of aerobic capability after LVRS.     

Dielectric behavior of pulmonary edema induced in the rat lung

The dielectric properties (conductivity, kappa and relative permittivity, epsilon) of excised rat lung are modified by lung air and water content. The measurements of these quantities were made over the frequency range of 10 kHz to 100 MHz with an open-ended coaxial probe. The following relationships were analyzed in an oleic acid-induced pulmonary edema model using 18 animals: the spectra of kappa, epsilon and the loss tangent as a function of lung air and water content. Secondly, an isolated-perfused lung system was produced to induce a gradual increase in lung water. The time course of kappa, epsilon and the loss tangent for one excised lung was analyzed. The principal findings were: (i) a decrease in kappa and epsilon with increasing air content, (ii) an increase in kappa and epsilon with increasing water content, and (iii) a good correlation between lung water content and maximum loss tangent that was insensitive to changes in air content. We conclude that this technique could provide a quantitative assessment of lung water during pulmonary edema formation.     

Chronologic changes of activities of naphthol AS-D acetate esterase and other nonspecific esterases in the mononuclear phagocytes of tuberculous lesions.

Nonspecific esterases of mononuclear phagocytes (MNs) were studied histochemically in the developing and healing tuberculous lesions produced in rabbit skin by bacille Calmette Guérin (BCG). Nonspecific esterases were assayed with the following substrates: naphthol AS-D acetate (AS-D), naphthol AS-D chloroacetate (AS-D Chl), naphthol AS acetate (AS) and alpha-naphthyl acetate (alpha-N), beta-Galactosidase, a lysosomal enzyme of MNs, was also assayed as a marker of MN activation. The number of MNs hydrolyzing AS-D Chl, AS, and alpha-N increased for 2 to 4 weeks after infection. These chronologic changes were similar to that of beta-galactosidase. In contrast, MNs hydrolyzing AS-D appeared predominantly in the healing lesions five to six weeks after infection. These MNs had the morphologic features of balloon-like cells. They contained few lysosomes and gathered in clumps far from the caseous center. The activity of the AS-D esterase was almost completely inhibited by various trypsin inhibitors, but not by the serine esterase inhibitor of phenylmethylsulfonyl-fluoride. These results suggest that the AS-D esterase is a trypsin-like esterase which participates in the healing of tuberculous lesions.     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid